A METHOD FOR DETERMINATION IN-SITU OF VARIATIONS WITHIN THE HEPATIC LOBULE OF HEPATOCYTE FUNCTION AND METABOLITE CONCENTRATIONS

A method is described for the production of detailed maps of intralobu lar variations of hepatocyte function and metabolite concentrations, b ased on variable destruction by digitonin of the lobule from the centr ilobular direction. Instead of the conventional approach, in which iso lated hepatocytes are then prepared and studied in suspension, perfusi on is continued after digitonin treatment and the function of the unaf fected lobular remnants is determined, or mean metabolite concentratio ns are measured by P-31-NMR. These measurements are plotted against th e degree of destruction, determined precisely after each study by auto mated quantitative histomorphometry. These plots are transformed into curves of the function or metabolite concentration of nominal single c ells at any point along the radius of the lobule. Gluconeogenesis from lactate remained stable, although reduced, even after 85-90% lobular destruction, predominated periportally and disappeared by 50% along th e radius of the lobule. In P-31-NMR studies, employing 1.5 mM lactate as substrate, narrowing of the intracellular P-i resonance was observe d as digitonin destruction increased; this was attributed to a decreas e in the intralobular heterogeneity of the intracellular pH, which fel l from approx. 7.9 to < 7.4 along the first 16% of the lobular radius (from the periportal end) and to < 7.3 in the remainder of the lobule. The ATP concentration rose, and then fell, along the radius of the lo bule in a centripetal direction. The method is potentially generally a pplicable to a wide range of hepatocellular functions and to the measu rement of metabolite concentrations, most conveniently those susceptib le to estimation by NMR.